Sensing Technology For Active Molecules Such As H₂O₂ Based On Microelectrode

As an important molecule in biological system, hydrogen peroxide playes an important role in many physiological processes. The adverse environmental conditions, such as drought, waterlogging, extreme temperatures and UV radiation would result in the activation of specific defense responses of plants including H2O2. Measurement of H2O2is helpful for understanding the defense responses in plants, screening and developing stress resistance plants. In cellular level, H2O2concentration rise in normal cells could induce cancer. Mounting evidence suggests that, compared with their normal counterparts, many types of cancer cell have increased levels of hydrogen peroxide. H2O2could help the cancer cells infiltrate and metastasize to other tissues and as an important signal molecule in cancer cells regulates of the entire process of tumor cell survival, proliferation and apoptosis. Tumor cells are strongly dependent on H2O2, also they are more sensitive to changes in H2O2concentration than normal cells. Therefore, increasing the H2O2concentration in cells by drugs is a method of inducing apoptosis of tumor cells. Understanding the changes of H2O2in the apoptosis of cancer by drugs has great significance in cancers’effective prevention and control.Hydrogen peroxide detection by chemiluminescence, fluorimetry and spectrophotometry are difficult for in vivo studying oxidative burst dynamically due to their requirement of tracers or instable chemical probes. Moreover, these methods have to be conducted in detached or ground plant samples, and the steps involved are complicated and time-consuming. Furthermore, detachment of a plant parts may result in stress and induce plant defence responses, including related defence gene expression and consecutive physiological processes. Furthermore, considering the dynamic change in H2O2levels during abiotic stresses in a field environment, continuous in vivo measurements are more favourable, but are infeasible when using optical probes.The electrochemical approach using microelectrode and ultramicroelectrode has attracted significant attention and has became an attractive method for in vivo and ex vivo biological application because of its high sensitivity, low cost, rapid response, compatibility for miniaturization, and compatibility with microfabrication technology. Based on electrochemistry, this thesis constructed different microelectrodes and ultramicroelectrodes based nano materials for in vivo detection of oxidative burst in plants and H2O2released from cancer cells. The main research content and results are shown as below:(1) Single-walled carbon nanotubes (SWCNTs) and hemoglobin (Hb) modified carbon fiber ultramicroelectrodes (CFUMEs) were employed to construct direct electron transfer based in vivo H2O2sensor. Hb remains high redox catalytic to H2O2reduction after being immobilized on SWCNTs coated CFUME. With the help of SWCNTs, the facilitated electron transfer of Hb effectively lowered the working potential to-0.1V, thus causing much less interference from other electrochemical active species without incorporating redox mediator or extra anti-interference coatings. The feature is especially preferred by in vivo sensing applications. The microbiosensor with high sensitivity and selectivity for H2O2is employed for real time continuously monitoring of oxidative burst on aloe for19.5hours.(2) SWCNTs and horseradish peroxidase (HRP)-room temperature ionic liquids (RTILs) modified carbon fiber ultramicroelectrodes (CFUMEs) were employed to construct direct electron transfer based ex vivo H2O2sensor. HRP remains high redox catalytic to H2O2reduction. The modified electrode has high affinity and sensitivity to H2O2, the detection limit is0.13uM. The sensor has less interference from other electrochemical active species without selectively permeable membranes. The sensor was used to detect the H2O2release from HeLa cells with AA or camptothecin, respectively.(3) In order to more comprehensive understand the concentration changes of different molecules in plants under stress, a field-compatible technique using electrochemical microbundle for real-time and simultaneous in vivo measurement of hydrogen peroxide, nitric oxide and pH was developed. Compared with the traditional optical probe method, the detection could be employed without ceasing plant growth or detaching any part of plant. Drought-stressed oilseed rape was used as a model system. When drought stress was applied, two large peaks in the NO level were observed at11h and22h, corresponding to3.1μM and2.6μM, respectively. In the presence of drought stress, the H2O2level increased from20.8h until the end of the measurement. The H2O2concentration increased to2.5μM after45h. The pH was observed to remain relatively stable during the first18h, and continuously increase during the remaining measurement time. The overall increase in pH in an oilseed rape stem was about1.08throughout the measurement. Indeed, the real-time measurements can be conducted in various stages of plant since the diameter of microbundle is less than1mm. Furthermore, this technique is promising to be used in screening plant resistance capability due to its low cost, simple operation and rapidity. This technique is also expected to help to understand the interaction mechanism of NO, H2O2and pH in stress signaling.(4) SWCNTs and Hb modified Pt microelectrodes were employed to construct direct electron transfer based in vivo H2O2sensor. The sensor was applied to detect the differences of oxidative burst between the resistant-drought type (RT) and wild type (WT) oilseed rapes. With drought stress, in WT oilseed rape, oxidative burst can be observed starting from7.8h and resulted in two large peaks at10.3h and26.5h, respectively. Compared to WT, RT oilseed rape showed only one oxidative burst starting from10.7h and resulting one large peak at15.8h during the40h measurement. RT showed lower H2O2level under drought stress, which may because of higher activities of superoxide dismutase (SOD) and enzymes related to hydrogen peroxide removal. The lower hydrogen peroxide accumulated in RT may thus explain the enhanced percent survival of RT oilseed rape under drought stress.